All About Alternative Splicing

This article was review by Thomas Cooper, MD from Baylor College of Medicine.

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What Is Alternative Splicing?

Alternative splicing is a variation of the constitutive splicing process by which noncoding pre-mRNA segments (introns) are removed from a transcript.¹ Unlike constitutive splicing, where exons are joined in a fixed order, alternative splicing selectively includes or excludes certain coding segments called exons, resulting in different mRNA transcripts and increasing the coding potential of eukaryotic genomes.² 

Alternative splicing enables a single gene to encode multiple mRNA variants and, consequently, diversifies protein isoform products. The advent of deep sequencing technologies has revealed that 95 percent of human genes undergo alternative splicing, often in multiple regions along their sequence.¹ This explains how approximately 20,000 human protein coding genes can produce over 90,000 distinct proteins.²

Alternative splicing and molecular research

Alternative splicing is crucial to gene expression, influencing protein functions such as binding interactions, localization, and enzymatic activity.² The regulation of alternative splicing is vital for normal tissue function and disruptions are linked to numerous diseases, including cancer.²

Despite its significance, alternative splicing’s global impact on cellular homeostasis and intricate molecular mechanisms remains incompletely characterized. Research into splicing mechanisms and patterns is essential for decerning fundamental biological principles, as well as clarifying the molecular basis of many diseases and developing new therapies.²

Alternative Splicing Mechanisms of Action

A complex splicing network interacts with epigenetic, transcriptional, and posttranscriptional controls to shape gene expression.³ This network is driven by multiple splicing mechanisms, including factors that autoregulate and cross-regulate each other, collectively influencing splicing activity and generating a dynamic web of regulatory inputs.³

What is the spliceosome?

At the core of the splicing process is the spliceosome, a highly intricate molecular complex responsible for removing introns from pre-mRNA.¹ Composed of small nuclear RNA (snRNA) that are assembled into core ribonucleoproteins (snRNPs), and over 300 auxiliary proteins, the spliceosome forms a megadalton complex—one of the most elaborate macromolecular structures in eukaryotic cells.^1,4

Splicing is closely linked with transcription, involving a dynamic interaction between the mRNA, RNA polymerase II (Pol II), and chromatin structures.⁵ “There's strong evidence that splicing of pre-mRNAs from most genes and presumably alternative splicing is co-transcriptional; it's all happening in a coordinated fashion,” explained Thomas Cooper, a molecular and cellular researcher at Baylor College of Medicine who investigates alternative splicing regulatory mechanisms and consequences in neuromuscular disease. 

During pre-mRNA transcription by Pol II, the spliceosome catalyzes two transesterification reactions along the RNA molecule, cleaving it twice and covalently joining adjacent exons, effectively excising an intron.^1,4 Sixteen different splicing factors are recruited at various stages of the splicing cycle to ensure accurate mRNA processing into mature, functional transcripts.^6,7

What are regulatory proteins in alternative splicing?

Splicing-associated RNA binding proteins (RBP) primarily belong to two major families: heterogeneous nuclear ribonucleoproteins (hnRNP) and serine-arginine (SR) proteins.⁴ These proteins have opposing roles, with SR proteins generally enhancing splicing and hnRNP typically repressing it. The splicing function of these proteins is influenced by their location at exons or introns along the pre-mRNA.

Located throughout the pre-mRNA are short, cis-acting motifs consisting of five to six nucleotides that are binding sites for RBP, which act in trans.^1,8 “When an exon that is alternatively spliced undergoes regulation, the sequence motifs required are located close by on the pre-mRNA,” said Cooper. “Often, they will be clustered and usually be within the first 100 or 200 nucleotides within the introns. They can also be within the exon.”

RBP splicing factors are sequence-specific, binding to their cognate sequence motifs like a lock and key. “Because the binding pocket of the RBP matches the sequence motif, the RBP binds to the specific set of several nucleotides. Then the RNA binding protein communicates to the splicing machinery to include or not include the exon into the mRNA,” Cooper explained.

Regulatory protein activity is modulated by phosphorylation and dephosphorylation cycles, which are sensitive to physiological conditions like stress or temperature.¹ This suggests that RBP adjust splicing activity in response to environmental changes, potentially influencing biological rhythms and behaviors.¹

Cellular regulation of alternative splicing

The fit between a splice site and the spliceosome components that bind it influences splicing activity. Strong splice sites closely match consensus sequences and are efficiently recognized by the spliceosome, while weak or suboptimal splice sites may be skipped, leading to alternative splicing and different mature mRNA variants.¹ Alternative splice sites tend to be suboptimal allowing for modulation by RBPs.

Transcription elongation rates can influence exon inclusion or skipping.¹ The kinetic coupling model explains that slow elongation can promote inclusion of exons with weak splice sites, though this rate can also facilitate the binding of splicing suppressors leading to exon skipping.⁹

In addition to proteins, noncoding RNAs (ncRNAs), such as microRNA, long ncRNA, and small interfering RNA, can also regulate alternative splicing.^2,10 They modulate the expression of key splicing factors during development and cell differentiation, providing an additional layer of regulation to the splicing process.

At the posttranscriptional level, nonsense mediated decay (NMD) is a process closely linked to alternative splicing that allows cells to control protein production.¹ Alternative splicing will sometimes introduce a premature stop codon into an mRNA, which NMD will recognize as an mRNA that needs to be degraded. “It's a very common mechanism of downregulation,” explained Cooper. “A truncated protein might cause a lot of problems … Nonsense mediated decay is a mechanism to get rid of that RNA before it's translated into a protein.” 

Major Alternative Splicing Functions

Cell fate and organ differentiation through alternative splicing

Alternative splicing plays a pivotal role in defining cell phenotypes and physiology in multicellular eukaryotes, often distinguishing between cell types more accurately than gene expression.¹

During development, alternative splicing patterns characterize specific cell and tissue phenotypes.⁷ Proper regulation of splicing factors ensures accurate tissue-specific gene expression and cell differentiation.

Temporal control of alternative splicing is also crucial for developmental transitions.¹¹ Disruptions in these coordinated splicing networks can affect tissue and organ homeostasis. “There's hundreds of genes that normally switch by alternative splicing from a fetal protein isoform to an adult protein isoform,” explained Cooper. For example, Cooper’s work determined that errant alternative splicing regulation is linked to forms of myotonic dystrophy, in which the skeletal muscle tissue experiences arrested development.¹² “It's stuck expressing fetal protein isoforms that aren't sufficient for the correct function of adult tissues.” 

Conservation in evolution and species differentiation

Alternative splicing is conserved throughout the phylogenetic tree with the same exons being alternatively spliced across multiple species.¹³ Examples of shared alternative splicing events suggest their importance to biological function.

Alternative splicing also plays a fundamental role shaping the molecular and phenotypic diversity between species.² Higher eukaryotes, especially primates, exhibit a greater proportion of alternatively spliced genes compared to unicellular eukaryotes.^1,2 Primates also feature alternative splicing profiles that are more closely related to species identity than to specific organ types.¹

Alternative splicing in cancer and disease

Understanding the role of alternative splicing in disease sheds light on the molecular mechanisms underlying these conditions. Alternative splicing plays a critical role in cell proliferation and apoptosis, processes closely tied to cancer.⁸ Cancer cells often exhibit aberrant splicing patterns driven by mutations or altered expression of splicing machinery elements.¹⁴ A key challenge in cancer research is determining whether specific splicing events are drivers of malignancy or merely byproducts.²

Splicing defects are also implicated in various other medical conditions, such as autism spectrum disorder.⁴ For instance, mutations in the splicing factor RBFOX1 is linked to autism, where it disrupts synaptic transmission and membrane excitability, adversely affecting neurodevelopmental pathways.¹¹